Article pubs.acs.org/crt
Nile Blue Can Photosensitize DNA Damage through Electron Transfer Kazutaka Hirakawa,*,† Kazuhiro Ota,† Junya Hirayama,‡ Shinji Oikawa,‡ and Shosuke Kawanishi§ †
Department of Applied Chemistry and Biochemical Engineering, Graduate School of Engineering, Shizuoka University, Johoku 3-5-1, Naka-ku, Hamamatsu, Shizuoka 432-8561, Japan ‡ Department of Environmental and Molecular Medicine, Mie University School of Medicine, Edobashi 2-174, Tsu, Mie 514-8507, Japan § Department of Pharmaceutical Sciences, Faculty of Pharmaceutical Sciences, Suzuka University of Medical Science, Kishioka 1001-1, Suzuka, Mie 510-0293, Japan S Supporting Information *
ABSTRACT: The mechanism of DNA damage photosensitized by Nile blue (NB) was studied using 32P-5′-end-labeled DNA fragments. NB bound to the DNA strand was possibly intercalated through an electrostatic interaction. Photoirradiated NB caused DNA cleavage at guanine residues when the DNA fragments were treated with piperidine. Consecutive guanines, the underlined G in 5′-GG and 5′-GGG, were selectively damaged through photoinduced electron transfer. The fluorescence lifetime of NB was decreased by guanine-containing DNA sequence, supporting this mechanism. Single guanines were also slightly damaged by photoexcited NB, and DNA photodamage by NB was slightly enhanced in D2O. These results suggest that the singlet oxygen mechanism also partly contributes to DNA photodamage by NB. DNA damage photosensitized by NB via electron transfer may be an important mechanism in medicinal applications of photosensitizers, such as photodynamic therapy in low oxygen.
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INTRODUCTION Photosensitized DNA damage is a potentially important mechanism for photodynamic therapy (PDT), a less invasive treatment for cancer and some nonmalignant conditions.1−4 PDT involves the administration of a photosensitizer followed by exposure of the tissue to visible nonthermal light. When the photosensitizer is illuminated with light of an appropriate wavelength, the photoexcited molecule induces photochemical damage to biomacromolecules such as DNA, resulting in cell death. In general, the most important mechanism of biomacromolecule damage is the generation of reactive oxygen species such as singlet oxygen (1O2) (type II mechanism).5−10 Photoinduced electron transfer from biomolecules to a photoexcited drug (type I mechanism)5 is also important.10−13 In certain cases, an inactive dye induces biomolecule photodamage. For example, rhodamine-6G, an analogue of xanthene dye, which is highly fluorescent and hardly induces photochemical reactions and/or 1O2 generation, photosensitizes DNA damage.14 Photosensitized biomolecule damage by an inactive dye may be useful for target-selective PDT because such a dye is safe under general conditions and hardly damages other biomolecules. However, rhodamine-6G cannot absorb long-wavelength light, the use of which is advantageous for PDT. For this purpose, Nile blue (NB, Figure 1), an inactive dye, might be a target-selective photosensitizer candidate because of its high absorptivity for the long-wavelength region (600−700 nm). Thus, in this study, DNA damage photosensitized by NB was examined, and its DNA-damaging activity was confirmed. NB is a benzophenoxazine compound and a © 2014 American Chemical Society
Figure 1. Structure of NB. The electrostatic potential map (right) was calculated by DFT at the B3LYP/6-31G* level. Red and blue indicate negative and positive charge, respectively.
well-known DNA-binding dye.15,16 These kinds of dyes exhibit relatively low systemic toxicity, and some of them retard tumor growth.17,18 The quantum yield of 1O2 generation (ΦΔ) by NB is negligibly small (0.005),19,20 and no phototoxic effect or photosensitized biomolecule damage has been reported. NB has the potential to demonstrate selective phototoxicity and may be useful for medicinal applications, including PDT.
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EXPERIMENTAL PROCEDURES
Materials. NB and the synthesized oligonucleotides containing only the adenine−thymine sequence (AATT: d(AAAATTTTAAAATTTT)2) and the guanine-containing sequence (AGTC: d(AAGCTTTGCAAAGCTT)2) were purchased from Sigma-Aldrich (St. Louis, MO, USA). These oligonucleotides were annealed for 5 min at 90 °C in 100 mM sodium phosphate buffer and gradually cooled at room temperature. As previously reported, the duplex DNA Received: December 26, 2013 Published: February 27, 2014 649
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could be prepared through this procedure.21,22 Restriction enzymes (AvaI and PstI) and T4 polynucleotide kinase were purchased from New England Biolabs (Beverly, MA, USA). [γ-32P]-ATP (222 TBq/ mmol) was from New England Nuclear (Boston, MA, USA). Riboflavin was from Wako Pure Chemicals, Ltd. (Osaka, Japan). Calf thymus DNA was from Sigma Chemical Co. (St. Louis, MO, USA). Preparation of 32P-5′-End-Labeled DNA Fragments. DNA damage photosensitized by NB was examined using 32P-labeled DNA fragments obtained from human genes. A singly labeled 337 bp fragment (PstI 2345−AvaI* 2681) was prepared from plasmid pbcNI, which carries a 6.6 kb BamHI chromosomal DNA segment containing the human c-Ha-ras-1 protooncogene, according to a method described previously.23 Nucleotide numbering starts with the BamHI site.24 The asterisk indicates 32P-labeling. Detection of DNA Damage Induced by Photosensitization of Nile Blue. The standard reaction mixture in a microtube (1.5 mL Eppendorf) contained NB, a 32P-labeled DNA fragment ( thymine (1.90 V) ≈ cytosine (1.90 V).30−32 Thus, the Gibbs free energy (ΔG) of the electron transfer was roughly estimated using the following equation ΔG = (E + − E−) − E(S1)
(5)
where E(S1) is the S1 energy estimated from the fluorescence maximum (669 nm), E+ is the oxidation potential of the guanine (1.24 V vs SCE),30−32 and E− is the reduction potential of NB (−0.46 V vs SCE).33 The estimated value of ΔG (−0.15 eV) indicates that electron transfer from guanine to the photoexcited NB is possible in terms of energy. Furthermore, molecular orbital (MO) calculations have revealed that 5′-G in the GG sequence in double-stranded DNA significantly lowers the highest occupied MO (HOMO) energy.34,35 Therefore, the cation radical on the 5′-G in the GG sequence arises either from the initial electron abstraction of this guanine by photoexcited NB or through charge migration from a relatively distant one-electron-oxidized nucleobase.30,36−39 The Φrf was decreased in the presence of GC-containing DNA, suggesting the quenching of S1 of NB by guanines. The measurement of τf of NB also supported the quenching of NB S1 by guanine residues. The estimated electron transfer rate constant (1.0 × 1010 s−1) was sufficiently large. The mechanism of DNA damage photosensitized by NB is proposed, as shown in Figure 8 and the Supporting
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CONCLUSIONS
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ASSOCIATED CONTENT
NB interacted with the DNA strand and photosensitized DNA damage at guanine residues. Consecutive guanines were damaged by photoexcited NB. The proposed main mechanism of this DNA damage is an electron transfer from the DNA base to the photoexcited NB. Because the quantum yield of the triplet formation of NB is small, the electron transfer from DNA to the S1 of NB should be important. In addition, the 1O2 mechanism partly contributes to DNA photodamage by NB. The electron-transfer mechanism is important for PDT in low oxygen.48 NB has an absorption band around 650 nm, which is an advantageous wavelength region for PDT. Because NB is not a photochemically active compound, NB should photosensitize only the binding biomolecule. These characteristics of NB may be useful in a DNA-selective photosensitizer.
Figure 8. Proposed mechanism of DNA damage photosensitized by NB.
Information. The major process of this DNA damage is mediated by the photoinduced electron transfer from the nucleobase to photoexcited NB, resulting in the formation of guanine cation radicals (dGuo•+) and neutral radicals of NB (NB•). The formed dGuo•+ may react with water molecules to form the C-8 OH adduct radical (dGuo−OH•).36,40 This radical may be converted by a reducing process into a 2,6diamino-4-hydroxy-5-formamidopyrimidine (FapyGua) residue, a piperidine-labile product.36,40 The reverse electron transfer from the NB• bound to the DNA strand to the dGuo− OH• might enhance this process. A similar mechanism was proposed in the case of DNA damage photosensitized by rhodamine-6G.14 However, competitive oxidation, which may
S Supporting Information *
Electron transfer rate constant, 8-oxodGuo formation induced by photosensitization of nile blue, and proposed scheme of 653
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(12) Hirakawa, K., Yoshida, M., Oikawa, S., and Kawanishi, S. (2003) Base oxidation at 5′ site of GG sequence in double-stranded DNA induced by UVA in the presence of xanthone analogues: Relationship between the DNA-damaging abilities of photosensitizers and their HOMO energies. Photochem. Photobiol. 77, 349−355. (13) Hirakawa, K., Fukunaga, N., Nishimura, Y., Arai, T., and Okazaki, S. (2013) Photosensitized protein damage by dimethoxyphosphorus(V) tetraphenylporphyrin. Bioorg. Med. Chem. Lett. 23, 2704−2707. (14) Hirakawa, K., Ochiai, S., Oikawa, S., and Kawanishi, S. (2011) Oxygen-independent DNA damage photosensitized by rhodamine-6G. Trends Photochem. Photobiol. 13, 29−35. (15) Chen, Q., Li, D., Yang, H., Zhu, Q., Xu, J., and Zhao, Y. (1999) Interaction of a novel red-region fluorescent probe, nile blue, with DNA and its application to nucleic acids assay. Analyst 124, 901−907. (16) Mitra, R. K., Sinha, S. S., and Pal, S. K. (2008) Interactions of nile blue with micelles, reverse micelles and a genomic DNA. J. Fluoresc. 18, 423−432. (17) Morgan, J., Potter, W. R., and Oseroff, A. R. (2000) Comparison of photodynamic targets in a carcinoma cell line and its mitochondrial DNA-deficient derivative. Photochem. Photobiol. 71, 747−757. (18) Singh, G., Espiritu, M., Shen, X. Y., Hanlon, J. G., and Rainbow, A. J. (2001) In vitro induction of PDT resistance in HT29, HT1376 and SK-NMC cells by various photosensitizers. Photochem. Photobiol. 73, 651−656. (19) Wainwright, M., Mohr, H., and Walker, W. H. (2007) Phenothiazinium derivatives for pathogen inactivation in blood products. J. Photochem. Photobiol., B 86, 45−58. (20) Hirakawa, K. (2009) Fluorometry of singlet oxygen generated via a photosensitized reaction using folic acid and methotrexate. Anal. Bioanal. Chem. 393, 999−1005. (21) Hirakawa, K., Hirano, T., Nishimura, Y., Arai, T., and Nosaka, Y. (2012) Dynamics of singlet oxygen generation by DNA-binding photosensitizers. J. Phys. Chem. B 116, 3037−3044. (22) Hirakawa, K., Nishimura, Y., Arai, T., and Okazaki, S. (2013) Singlet oxygen generating activity of an electron donor-connecting porphyrin photosensitizer can be controlled by DNA. J. Phys. Chem. B 117, 13490−13496. (23) Yamamoto, K., and Kawanishi, S. (1991) Site-specific DNA damage induced by hydrazine in the presence of manganese and copper ions. The role of hydroxyl radical and hydrogen atom. J. Biol. Chem. 266, 1509−1515. (24) Capon, D. J., Chen, E. Y., Levinson, A. D., Seeburg, P. H., and Goeddel, D. V. (1983) Complete nucleotide sequences of the T24 human bladder carcinoma oncogene and its normal homologue. Nature 302, 33−37. (25) Maxam, A. M., and Gilbert, W. (1980) Sequencing end-labeled DNA with base-specific chemical cleavages. Methods Enzymol. 65, 499−560. (26) Benesi, H. A., and Hildebrand, J. H. (1949) A spectrophotometric investigation of the interaction of iodine with aromatic hydrocarbons. J. Am. Chem. Soc. 71, 2703−2707. (27) Pyle, A. M., Rehman, J. P., Meshoyrer, R., Kumar, C. V., Turro, N. J., and Barton, J. K. (1989) Mixed-ligand complexes of ruthenium(II): Factors governing binding to DNA. J. Am. Chem. Soc. 111, 3051−3058. (28) Ogilby, P. R., and Foote, C. S. (1983) Chemistry of singlet oxygen. 42. Effect of solvent, solvent isotopic substitution, and temperature on the lifetime of singlet molecular oxygen (1Δg). J. Am. Chem. Soc. 105, 3423−3430. (29) Ito, K., Inoue, S., Yamamoto, K., and Kawanishi, S. (1993) 8Hydroxydeoxyguanosine formation at the 5′ site of 5′-GG-3′ sequences in double-stranded DNA by UV radiation with riboflavin. J. Biol. Chem. 268, 13221−13227. (30) Lewis, F. D., and Wu, Y. (2001) Dynamics of superexchange photoinduced electron transfer in duplex DNA. J. Photochem. Photobiol., C 2, 1−16. (31) Seidel, C. A. M., Schulz, A., and Sauer, M. H. M. (1996) Nucleobase-specific quenching of fluorescent dyes. 1. Nucleobase one-
guanine damage photosensitized by NB. This material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*Tel/Fax: +81-53-478-1287. E-mail:
[email protected]. jp. Funding
This research was funded by JSPS KAKENHI grant no. 23750186. Notes
The authors declare no competing financial interest.
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ABBREVIATIONS APN, absorbed photon number; bp, base pair; DFT, density functional theory; dGuo, 2′-deoxyguanosine; dGuo•+, guanine cation radicals; dGuo−OH·, C-8 OH adduct radical; FapyGua, 2,6-diamino-4-hydroxy-5-formamidopyrimidine; ΔG, Gibbs free energy; HOMO, highest occupied molecular orbital; HPLC-ECD, electrochemical detector coupled to highperformance liquid chromatography; MO, molecular orbital; NB, nile blue; NB•, neutral radicals of NB; 1O2, singlet oxygen; PDT, photodynamic therapy; 8-oxodGuo, 8-oxo-7,8-dihydro2′-deoxyguanosine; S1, singlet excited state; SCE, saturated calomel electrode; ΦD, relative quantum yield of DNA damage; Φrf, relative fluorescence quantum yield; ΦΔ, quantum yield of 1 O2 generation; τf, fluorescence lifetime
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REFERENCES
(1) Dolmans, D. E., Fukumura, D., and Jain, R. K. (2003) Photodynamic therapy for cancer. Nat. Rev. Cancer 3, 380−387. (2) Castano, A. P., Mroz, P., and Hamblin, M. R. (2006) Photodynamic therapy and anti-tumour immunity. Nat. Rev. Cancer 6, 535−545. (3) Wilson, B. C., and Patterson, M. S. (2008) The physics, biophysics and technology of photodynamic therapy. Phys. Med. Biol. 53, R61−R109. (4) Collins, H. A., Khurana, M., Moriyama, E. H., Mariampillai, A., Dahlstedt, E., Balaz, M., Kuimova, M. K., Drobizhev, M., Yang, V. X. D., Phillips, D., Rebane, A., Wilson, B. C., and Anderson, H. L. (2008) Blood-vessel closure using photosensitizers engineered for two-photon excitation. Nat. Photonics 2, 420−424. (5) Foote, C. S. (1991) Definition of type I and type II photosensitized oxidation. Photochem. Photobiol. 54, 659. (6) DeRosa, M. C., and Crutchley, R. J. (2002) Photosensitized singlet oxygen and its applications. Coord. Chem. Rev. 233−234, 351− 371. (7) Schweitzer, C., and Schmidt, R. (2003) Physical mechanisms of generation and deactivation of singlet oxygen. Chem. Rev. 103, 1685− 1758. (8) Lang, K., Mosinger, J., and Wagneroviá, D. M. (2004) Photophysical properties of porphyrinoid sensitizers non-covalently bound to host molecules; models for photodynamic therapy. Coord. Chem. Rev. 248, 321−350. (9) Jarvi, M. T., Patterson, M. S., and Wilson, B. C. (2012) Insights into photodynamic therapy dosimetry: Simultaneous singlet oxygen luminescence and photosensitizer photobleaching measurements. Biophys. J. 102, 661−671. (10) Kawanishi, S., Hiraku, Y., and Oikawa, S. (2001) Damage by oxidative stress and its role in carcinogenesis and aging. Mutat. Res. 488, 65−76. (11) Boone, E., and Schuster, G. B. (2002) Long-range oxidative damage in duplex DNA: The effect of bulged G in a G-C tract and tandem G/A mispairs. Nucleic Acids Res. 30, 830−837. 654
dx.doi.org/10.1021/tx400475c | Chem. Res. Toxicol. 2014, 27, 649−655
Chemical Research in Toxicology
Article
electron redox potentials and their correlation with static and dynamic quenching efficiencies. J. Phys. Chem. 100, 5541−5553. (32) Chirvony, V. S., Galievsky, V. A., Kruk, N. N., Dzhagarov, B. M., and Turpin, P.-Y. (1997) Photophysics of cationic 5,10,15,20-tetrakis(4-N-methylpyridyl)porphyrin bound to DNA, [poly(dA-dT)]2 and [poly(dG-dC)]2: On a possible charge transfer process between guanine and porphyrin in its excited singlet state. J. Photochem. Photobiol., B 40, 154−162. (33) Pheeney, C. G., and Barton, J. K. (2013) Intraduplex DNAmediated electrochemistry of covalently tethered redox-actie reporters. J. Am. Chem. Soc. 135, 14944−14947. (34) Sugiyama, H., and Saito, I. (1996) Theoretical studies of GGspecific photocleavage of DNA via electron transfer: Significant lowering of ionization potential and 5′-localization of HOMO of stacked GG bases in B-form DNA. J. Am. Chem. Soc. 118, 7063−7068. (35) Yoshioka, Y., Kitagawa, Y., Takano, Y., Yamaguchi, K., Nakamura, T., and Saito, I. (1999) Experimental and theoretical studies on the selectivity of GGG triplets toward one-electron oxidation in B-form DNA. J. Am. Chem. Soc. 121, 8712−8719. (36) Burrows, C. J., and Muller, J. G. (1998) Oxidative nucleobase modifications leading to strand scission. Chem. Rev. 98, 1109−1151. (37) Hall, D. B., Holmlin, R. E., and Barton, J. K. (1996) Oxidative DNA damage through long-range electron transfer. Nature 382, 731− 735. (38) Nunez, M. E., Hall, D. B., and Barton, J. K. (1999) Long-range oxidative damage to DNA: Effects of distance and sequence. Chem. Biol. 6, 85−97. (39) Douki, T., Angelov, D., and Cadet, J. (2001) UV laser photolysis of DNA: effect of duplex stability on charge-transfer efficiency. J. Am. Chem. Soc. 123, 11360−11366. (40) Ravanat, J.-L., Douki, T., and Cadet, J. (2001) Direct and indirect effects of UV radiation on DNA and its components. J. Photochem. Photobiol., B 63, 88−102. (41) Kasai, H., Yamaizumi, Z., Berger, M., and Cadet, J. (1992) Photosensitized formation of 7,8-dihydro-8-oxo-2′-deoxyguanosine (8hydroxy-2′-deoxyguanosine) in DNA by riboflavin: A non singlet oxygen mediated reaction. J. Am. Chem. Soc. 114, 9692−9694. (42) Steenken, S., Jovanovic, S. V., Bietti, M., and Bernhard, K. (2000) The trap depth (in DNA) of 8-oxo-7,8-dihydro-2′deoxyguanosine as derived from electron-transfer equilibria in aqueous solution. J. Am. Chem. Soc. 122, 2373−2374. (43) Lim, K. S., Taghizadeh, K., Wishnok, J. S., Babu, I. R., Shafirovich, V., Geacintov, N. E., and Dedon, P. C. (2012) Sequencedependent variation in the reactivity of 8-oxo-7,8-dihydro-2′deoxyguanosine toward oxidation. Chem. Res. Toxicol. 25, 366−373. (44) Cadet, J., Berger, M., Buchko, G. W., Joshi, P. C., Raoul, S., and Ravanat, J.-L. (1994) 2,2-Diamino-4-[(3,5-di-O-acetyl-2-deoxy-β-Derythropentofuranosyl) amino]-5-(2H)-oxazolone: A novel and predominant radical oxidation product of 3′,5′-di-O-acetyl-2′-deoxyguanosine. J. Am. Chem. Soc. 116, 7403−7404. (45) Raoul, S., Berger, M., Buchko, G. W., Joshi, P. C., Morin, B., Weinfeld, M., and Cadet, J. (1996) 1H, 13C and 15N nuclear magnetic resonance analysis and chemical features of the two main radical oxidation products of 2′-deoxyguanosine: Oxazolone and imidazolone nucleosides. J. Chem. Soc., Perkin Trans. 2, 371−381. (46) Ravanat, J.-L., Di Mascio, P., Martinez, G. R., Medeiros, M. H., and Cadet, J. (2000) Singlet oxygen induces oxidation of cellular DNA. J. Biol. Chem. 275, 40601−40604. (47) Hirakawa, K., Kawanishi, S., and Hirano, T. (2005) The mechanism of guanine specific photo-oxidation in the presence of berberine and palmatine: Activation of photosensitized singlet oxygen generation through DNA-binding interaction. Chem. Res. Toxicol. 18, 1545−1552. (48) Bratasz, A., Kulkarni, A. C., and Kuppusamy, P. (2007) A highly sensitive biocompatible spin probe for imaging of oxygen concentration in tissues. Biophys. J. 92, 2918−2925.
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